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E2 Aptamer
E2 Aptamer
Han Yue Zheng, Omar A. Alsager, Cameron S. Wood, Justin M. Hodgkiss, and Natalie O. V. Plank
06F904-1 J. Vac. Sci. Technol. B 33(6), Nov/Dec 2015 2166-2746/2015/33(6)/06F904/8/$30.00 C 2015 American Vacuum Society
V 06F904-1
06F904-2 Zheng et al.: CNT FET aptasensors for estrogen detection in liquids 06F904-2
current.12,25,32 We have observed a measurable current CNT FET was then baked at a 200 C on a hot plate in air.
change on exposure to E2 for the 35-mer E2 CNT FET apta- Scanning electron microcopy (SEM) via the Jeol JSM 6500F
sensor, but no E2 response is observed for the 75-mer E2 was used to determine the morphology of the CNTs in the
aptamer. We also tested a control device with a random 35- device platform. The CNT FETs are then ready for chemical
mer DNA sequence, which shows no E2 specific response. functionalization to fabricate aptasensors.
DNA Sequence
35-mer E2 5 -NH2-AAGGGATGCCGTTTGGGCCCAAGTTCGGCATAGTG-30
0
FIG. 1. (Color online) (a) SEM image shows uniform CNT film on SiO2/Si substrates. (b) A schematic diagram of CNT FET measured in liquid using Ag/
AgCl as reference electrodes. (c) An overview optical image of CNT FET post SU8 electrode encapsulation: the areas of CNTs exposed for aptamer function-
alization are indicated by the rectangular solid lines.
solution if required. To test the devices in a liquid environ- shown in Fig. 1(b). Figure 1(c) shows an optical microscope
ment, a handmade PDMS well was placed on top of the image of the electrodes post SU8 encapsulation. The exposed
CNT FET to avoid liquid leakage to the probe electrodes. CNT area marked by the rectangle is 100 10 lm where those
Buffer solution of 150 ll was added into PDMS well before CNTs are the active area for aptamer functionalization.
measurement to ensure that the exposed active area of the The transfer characteristics of a CNT FET are shown in
reference electrode, which is enclosed in plastic up to the Fig. 2 for both the case of a back gated CNT FET through the
end pad, was completely covered in buffer at all times to pre- SiO2 and a liquid gated CNT FET. As expected, the CNT
vent the issue of an altered I-V response coming from a FETs exhibit p-type semiconductor behavior. In back gating,
change in the active area of the gate electrode. All electrical the voltage Vbg is applied through the highly doped Si, which
measurements were taken with the Agilent 4156C parameter acts as the electrode, and the 100 nm thermal SiO2 is an effec-
analyzer connected to the sample via micromanipulators and tive dielectric. The voltage Vbg is swept from þ10 to 10 V
a Rucker and Kolls probes station. During real time sensing and a fixed Vds ¼ 100 mV. The back gated device shows poor
measurements, the applied voltage and time are set from pa- field dependency with undefined Vth. In liquid gating, the volt-
rameter analyzer. The gate voltage was set to 0, the source–- age Vlg is applied through the Ag/AgCl reference electrode.
drain voltage was set to 50 or 100 mV, and the time step was Charges within the PBS buffer redistribute due to the applied
1 s. Throughout the sensing measurements, 10 ll of the E2 voltage, and an electric double layer (EDL) is formed, which
analyte was added. The concentration of E2 in PDMS varies acts as the dielectric region.32,34 The transfer characteristics
from 50 nM, 147 nM, 640 nM, and 1.6 lM. achieved from the same CNT FET show much stronger field
dependency properties under liquid gating compared to back
III. RESULTS AND DISCUSSION gating, even at a shorter sweeping voltage range from þ0.5 to
0.5 V and a fixed Vds ¼ 50 mV. Moreover, for the liquid
A. Device platform gated device, there is a clearly defined Vth ¼ þ0.1 V. To oper-
Uniform films of CNTs have been successfully realized on ate the CNT FET as a biosensor for E2, it is advantageous to
the 2-thiolpyridine functionalized SiO2/Si substrates [Fig. 1(a)] operate the device under a liquid gate environment. Figure 2
via the CNT DCB suspension method detailed in Sec. II. In order clearly shows that these network CNT FETs exhibit suitable
to use CNT FETs as sensor platforms, the CNT FET electrodes device performance.
are encapsulated with SU8 to prevent leakage currents from the
reference electrode and the source and drain, as schematically B. Aptamer immobilization
In order to immobilize aptamers on the CNT FET surfaces,
it is necessary to functionalize the pristine CNTs, as described
in Sec. II. PBASE is chosen to act as the linker between the
CNT and the aptamer because it provides nondestructive func-
tionalization of the CNT surface, meaning that the structure
and hence the electronic properties of the CNTs are not dam-
aged. The aptamer used in this research is functionalized by
amine groups (-NH2) at the 50 end. In our experiments, the
CNT FETs are first immersed in PBASE methanol solu-
tion.12,35 PBASE stacks onto the CNT sidewalls due to the
p–p interaction between the pyrene on PBASE and the ben-
zene rings on CNTs, as shown schematically in Fig. 3(a).
Once the PBASE functionalized CNT FET is immersed in the
aptamer/EDC/NHS solution for 3 h, the amine groups on the
50 end of the aptamer are then linked onto PBASE by nucleo-
philic substitution and formation of an amide bond (OC-NH)
FIG. 2. (Color online) Transfer characteristics of the same CNT FET using
back gating (Vds ¼ 100 mV) and liquid gating (Vds ¼ 50 mV) after SU8 elec- between the aptamer and PBASE.35 The NH2 terminated
trode encapsulation. aptamers are the most widely available form used for
FIG. 3. (Color online) Schematic representation of the CNT FET functionalization process: (a) PBASE stacks onto the CNT surfaces via p–p interaction. (b)
Aptamers are tethered on to the PBASE coated CNTs, via nucleophilic substitution. (c) When the aptamer senses the E2, the DNA strand is wrapped around
the analyte, bringing charges closer to the CNT surface.
conjugation and aptasensor research.36 Primary amines on Tween20 passivation), and the same CNT FET post E2 response
base groups of DNA as part of aromatic rings are not suffi- are all compared under liquid gating in 0.05 PBS (Fig. 4).
ciently reactive for controlled binding, unlike the alkyl amine It is clear that after the CNT FET has been functionalized
added to the end of the aptamer.37 Therefore, the alkyl amine with the E2 aptamer and Tween20 passivation, the magni-
terminated aptamer on the 50 end is necessary to link onto the tude of the current is reduced in comparison to the pristine
N-hydroxysuccinimide group of PBASE to form the amide CNT FET. The reduction in current is mainly due to the
tether.35 Without the NH2 termination, the aptamers will Tween20 passivation, which can act as scattering centers on
adsorb nonselectively onto the CNTs due to the p–p interac- the CNT surface and has the effect of both hindering the
tion between the benzene ring on CNT sidewall and the purine hole transportation along the CNTs and influencing the tube
and pyrimidine bases on aptamer.38 However, with the NH2 to tube junction sites in the percolating network of CNTs.
termination, the aptamer linking is controlled, and it takes After exposure to 1.6 lM of the E2 analyte, the functional-
place on the PBASE, as schematically shown in Fig. 3(b). To ized CNT FET device shows an increase in current (Fig. 4),
avoid hydrolysis of the PBASE during the aptamer functional- which can be explained by the negatively charged molecules
ization in liquid, the aptamer solution is prepared in a buffer of the DNA aptamer moving closer to CNT surface and
containing the EDC/NHS coupling solution. This ensures that changing the effective gating of the device.34 During the liq-
EDC/NHS activates the carboxyl groups if hydrolysis does uid gate measurements of the CNT FETs, an EDL is formed
happen.1,15 After the aptamer immobilization, the entire CNT close to the CNT surface. The thickness of the EDL is the
FET device chips are submerged in 0.05 wt. % Tween20 to Debye length.25,32,34 In CNT FET aptasensors, the confor-
passivate any remaining unfunctionalized CNTs and the mational change of negatively charged aptamers after bind-
exposed SiO2 substrate to avoid nonspecific adsorption of the ing alters the EDL at the CNT surface. For the binding
target analyte during the sensing measurements.28,30 events that take place within the Debye length, a change in
Aptamers are negatively charged due to the phosphate the current response will be observed for the CNT FET due
groups of the DNA backbone. After recognition between the to the effective change to the gate potential. Thus, the length
E2 aptamer and the E2 target analyte, the conformation of of recognition element prior to and upon detection is critical.
the aptamer will change and it may appear as though the Using the transfer characteristics of the CNT FETs (Fig.
aptamer “latches on” to the E2 molecule [Fig. 3(c)]. The 4), we have been able to determine that the E2 molecule has
change in conformation of the aptamer from random coil altered the current of the device. Previously, Pacios et al.15
motion in the solution to the aptamer wrapping onto the tar- have observed changes in the CNT FET transfer characteris-
get brings the negative charges from the DNA closer to the tics for a thrombin aptamer functionalized CNT FET to
CNT surface. Various degrees of folding are expected for
the unbound aptamer; however, the more compact conforma-
tion associated with E2 binding means that the negative
charges from the DNA will, on average, be closer to the sur-
face of the CNTs than in the unbound state. The binding
induced more compact conformation has been demonstrated
by previous studies using this aptamer tethered to nanopar-
ticles1 or electrode surfaces.5 The conformational change of
the aptamer structure also alters the charge distribution in
the EDL.1,15,25,32 The subsequent screening effect due to
charge redistribution has a large impact on the current
response of the CNT FET, and it is this effect that we aim to
observe in real time for the devices.
C. Electrical measurement FIG. 4. (Color online) Liquid gate performance of a pristine CNT FET, the
same CNT FET functionalized with the 35-mer E2 aptamer and 0.05 wt. %
1. Transfer characteristics Tween20 passivation, and the same CNT FET postexposure to 1.6 lM con-
centration of E2. The measurement is swept from þ1 V to 500 mV at fixed
The transfer characteristics for a bare CNT FET, the same Vds ¼ 100 mV. The device is measured in 0.05 PBS using an Ag/AgCl ref-
CNT FET functionalized with the 35-mer E2 aptamer (with erence electrode as the gate.
Sensor platform Analytical techniques Detection limit (M) Dynamic range (M) Real time Year and references
ACKNOWLEDGMENTS
H.Z. thanks Victoria University of Wellington and the
FIG. 7. (Color online) Normalized current change (DI/I0) in response to the Chinese Government joint Scholarship. N.P. thanks the
added concentration of the E2 analyte for the 35-mer E2 aptamer CNT FET, Foundation for Research Science and Technology Project
the random DNA sequence CNT FET, and the 35-mer E2 aptamer CNT
FET response to PBS buffer.
No. VICX0911 and the Marsden fund, Project No. E1728.
O.A. acknowledges a fellowship from King Abdulaziz City
comparison to the 35-mer E2 aptamer CNT FET. Although for Science and Technology.
there is a small increase in current at 50 nM, there is no real 1
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